CN111505138B - Characteristic spectrum construction method and quality detection method of fructus alpiniae oxyphyllae formula granules - Google Patents
Characteristic spectrum construction method and quality detection method of fructus alpiniae oxyphyllae formula granules Download PDFInfo
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Abstract
The invention relates to a method for constructing a characteristic spectrum of fructus alpiniae oxyphyllae formula granules and a quality detection method thereof. The construction method comprises the following steps: preparation of control solutions: dissolving pomelo ketone and kaempferide reference substances in solvent to obtain reference substance solution; preparation of a test solution: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution; and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution. The characteristic spectrum constructed by the construction method can comprehensively reflect the characteristics of the fructus alpiniae oxyphyllae formula particles, particularly realizes the detection of volatile oil in the fructus alpiniae oxyphyllae formula particles, and can well control the quality of the fructus alpiniae oxyphyllae formula particles.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a characteristic spectrum construction method and a quality detection method of fructus alpiniae oxyphyllae formula granules.
Background
Fructus Alpinae Oxyphyllae (Alpinia Oxyphylla Miq) is dry mature fruit of perennial herb of Zingiberaceae, has warm nature and pungent taste, and has effects of invigorating spleen and kidney channels, warming spleen, relieving diarrhea, arresting saliva, warming kidney, stopping nocturnal emission, and reducing urination, and can be used for treating abdominal pain, vomiting, diarrhea, anorexia, excessive saliva, etc. caused by kidney qi deficiency and failure to arrest. Mainly produced in Hainan, Guangdong, Guangxi, etc., is one of the genuine medicinal materials in Hainan and also one of the four great south medicinal materials. Modern pharmacology indicates that fructus alpiniae oxyphyllae has various pharmacological effects, and meanwhile, more and more researches indicate that the main components of the fructus alpiniae oxyphyllae playing the drug effect are volatile oil: as people think of Luoqin and the like, the volatile oil of fructus alpiniae oxyphyllae has obvious bacteriostatic action and has larger potential in the aspect of antibiosis; research of Liuchanghong iris and the like shows that the chemical component of the nootropic volatile oil, namely nootkatone, has a protective effect on chronic kidney injury and explains the kidney tonifying and urination reducing mechanism of the nootkatone; in addition, the active ingredients of the sharpleaf galangal fruit which have the effects of warming spleen and relieving diarrhea are volatile oil. In conclusion, the volatile oil component of fructus alpiniae oxyphyllae is an important component of fructus alpiniae oxyphyllae for exerting the drug effect.
The traditional Chinese medicine decoction is a prescription which is prepared for patients on the basis of dialectical treatment by a clinical traditional Chinese medicine doctor under the guidance of the traditional Chinese medicine theory, is continuously used up to now since commercial Yi creation decoction, and makes a great contribution to the medical health of Chinese nationality. But with the development of modern society and the improvement of technological level. The traditional Chinese medicine decoction cannot meet the fast-paced life style of modern people, and the traditional Chinese medicine formula granules are produced at the same time. The traditional Chinese medicine formula particle is a new clinical use form of pure traditional Chinese medicine prepared by taking traditional Chinese medicine decoction pieces which are processed by a modern processing technology and accord with the traditional processing specification as raw materials, and applying modern scientific technology to extraction, solid-liquid separation, concentration, drying, granulation and packaging; the functions, indications, properties and taste of the traditional Chinese medicine decoction pieces are consistent with those of the traditional Chinese medicine decoction pieces, the efficacy and the property of the traditional Chinese medicine decoction pieces are kept, and the traditional Chinese medicine decoction pieces have the characteristics of convenience for taking and dispensing of patients.
According to the regulation of the 2015 edition of Chinese pharmacopoeia, the content of the volatile oil in the sharpleaf galangal fruit decoction pieces is not less than 1.0%. However, the fructus alpiniae oxyphyllae volatile oil is unstable and volatile, so that most of the volatile oil is lost in the production and storage processes of the preparation, and the drug effect is influenced, therefore, the quality of the existing fructus alpiniae oxyphyllae formula particles is difficult to ensure to be equal to that of the traditional Chinese medicine decoction, particularly the content of the volatile oil in the fructus alpiniae oxyphyllae formula particles is difficult to achieve the drug effect equal to that of the traditional Chinese medicine decoction if the content of the volatile oil in the fructus alpiniae oxyphyllae formula particles is too low.
At present, the quality research on the fructus alpiniae oxyphyllae formula particles is less, and particularly, the volatile oil detection is carried out, so that a better quality standard is lacked to ensure the stable quality of the fructus alpiniae oxyphyllae formula particles. The method for acquiring comprehensive Chinese medicine chemical component characteristic information by utilizing the characteristic map is widely applied to the quality control of the Chinese medicine, and the Chinese medicine characteristic map expresses and reflects the integral chemical characteristics of the Chinese medicine, so that the quality of the Chinese medicine preparation can be effectively monitored, and the curative effect is improved and stabilized. Therefore, it is necessary to establish a characteristic map of the fructus alpiniae oxyphyllae formula granules.
Disclosure of Invention
Based on this, there is a need for a method for constructing a characteristic map of fructus alpiniae oxyphyllae formula granules. The characteristic spectrum constructed by the construction method can comprehensively reflect the characteristics of the fructus alpiniae oxyphyllae formula particles, particularly realizes the detection of volatile oil in the fructus alpiniae oxyphyllae formula particles, has comprehensive and reliable quality detection and short time, and can well control the quality of the fructus alpiniae oxyphyllae formula particles.
A method for constructing a characteristic spectrum of fructus alpiniae oxyphyllae formula granules comprises the following steps:
preparation of control solutions: dissolving pomelo ketone and kaempferide reference substances in solvent to obtain reference substance solution;
preparation of a test solution: adding an extraction solvent into the fructus alpiniae oxyphyllae formula particles for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;
detecting the reference solution and the test solution by using an ultra-high performance liquid chromatography, wherein the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;
the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;
the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;
and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.
In one embodiment, the conditions of the ultra high performance liquid chromatography include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 250-260 nm; flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
In one embodiment, the conditions of the ultra high performance liquid chromatography comprise:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 254 nm; flow rate: 0.3 mL/min; column temperature: at 30 ℃.
In one embodiment, the sample solution is prepared by using a methanol aqueous solution with a volume fraction of 45-55%.
In one embodiment, the method of extraction is ultrasonic extraction: the ultrasonic power is 200-300W, and the frequency is 20-30 kHZ; the extraction time is 25-35 min.
The invention also provides a quality detection method of the fructus alpiniae oxyphyllae formula granules, which comprises the following steps:
preparation of index component reference solution: dissolving a nootkatone reference substance in a solvent to prepare index reference substance solutions with different concentrations;
preparing a sample solution to be tested: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules to be detected for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test sample solution;
establishing a standard curve: detecting the index component reference substance solutions with different concentrations by using ultra-high performance liquid chromatography, and constructing a concentration-peak area standard curve of the index component;
and (3) detection: detecting the sample solution to be detected by using ultra-high performance liquid chromatography, and comparing a detection map with a concentration-peak area standard curve of the index component;
wherein, the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent to 0.15 percent, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;
the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;
the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;
and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.
In one embodiment, the conditions of the ultra high performance liquid chromatography comprise:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 250-260 nm; flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
In one embodiment, the conditions of the ultra high performance liquid chromatography comprise:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 254 nm; flow rate: 0.3 mL/min; column temperature: at 30 ℃.
In one embodiment, in the preparation of the sample solution to be tested, the extraction solvent is a methanol aqueous solution with a volume fraction of 45% to 55%.
In one embodiment, the extraction method is ultrasonic extraction, the ultrasonic power is 200-300W, and the frequency is 20-30 kHZ; the extraction time is 25-35 min.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method for constructing the characteristic spectrum of the sharpleaf galangal fruit formula particles takes ultra-high performance liquid chromatography (UPLC) as a technical means, adopts a proper mobile phase to carry out gradient elution, can obtain the characteristic spectrum of the sharpleaf galangal fruit formula particles, particularly detects volatile oil components in the sharpleaf galangal fruit formula particles, can comprehensively reflect main chemical component information of the sharpleaf galangal fruit formula particles, is comprehensive and reliable in quality detection, can accurately detect and evaluate the quality of the sharpleaf galangal fruit formula particles, and has important significance for ensuring the clinical curative effect of the sharpleaf galangal fruit formula particles. Meanwhile, the method has the advantages of simplicity, short time consumption, good stability, high precision and good reproducibility.
(2) The quality detection method provided by the invention can comprehensively reflect the main chemical component information of the fructus alpiniae oxyphyllae formula particles, can accurately measure the content of nootkatone in the fructus alpiniae oxyphyllae formula particles, is convenient for comprehensively monitoring the quality of the fructus alpiniae oxyphyllae formula particles, can synchronously perform qualitative and quantitative analysis, can accurately and effectively distinguish whether the volatile oil equivalent to the volatile oil in the fructus alpiniae oxyphyllae standard decoction is added in the fructus alpiniae oxyphyllae formula particles, and can ensure the safety, effectiveness and controllable quality of the medicine as a whole.
Drawings
FIG. 1 is a chromatogram of a control kaempferide;
FIG. 2 is a chromatogram of control nootkatone;
fig. 3 is a characteristic spectrum of the constructed sharpleaf galangal fruit formula granules;
FIG. 4 is a chromatogram of standard decoction of fructus Alpinae Oxyphyllae;
fig. 5 is a chromatogram of fructus alpiniae oxyphyllae formula granules prepared by a conventional process;
FIG. 6 is a chromatogram of a beta-cyclodextrin negative control solution in a methodological assay;
FIG. 7 is a chromatogram of a silica negative control solution in a methodological assay;
FIG. 8 is a chromatogram of a 50% methanol negative control solution in a methodological assay.
Detailed Description
The method for constructing the characteristic spectrum of the fructus alpiniae oxyphyllae formula particle and the quality detection method thereof are further described in detail with reference to specific examples.
The embodiment of the invention provides a method for constructing a characteristic spectrum of fructus alpiniae oxyphyllae formula granules, which comprises the following steps:
preparation of control solutions: dissolving reference substances of nootkatone and kaempferide in solvent to obtain reference substance solution;
preparation of a test solution: adding an extraction solvent into the fructus alpiniae oxyphyllae formula granules for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;
and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph for detection, wherein a mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, a mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, and the elution mode is gradient elution.
In one specific embodiment, the gradient elution comprises the following procedure:
the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;
the volume percentage of the mobile phase A is increased from 16% to 23% in 10-25 min;
increasing the volume percentage of the mobile phase A from 23% to 73% within 25-35 min;
and (3) reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min.
In one specific embodiment, the conditions of the ultra high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 250-260 nm; flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
More specifically, the conditions of the ultra high performance liquid chromatograph include:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; detection wavelength: 254 nm; flow rate: 0.3 mL/min; column temperature: at 30 ℃.
In one specific embodiment, the conditions of the ultra high performance liquid chromatograph further comprise: the theoretical plate number is not less than 100000 calculated according to the nootkatone peak.
In one specific embodiment, in the preparation of the test solution, the extraction solvent is a methanol aqueous solution with a volume fraction of 45% to 55%. Correspondingly, the extraction method can be ultrasonic extraction, and more specifically, the process conditions of the ultrasonic extraction include: the ultrasonic power is 200-300W, the frequency is 20-30 kHZ, and the extraction time is 25-35 min.
In one specific embodiment, the solvent used in the preparation of the control solution may be an alcohol solvent, preferably methanol. More specifically, the concentrations of the nootkatone and the kaempferide in the reference solution are 105-115 mu g/mL and 80-90 mu g/mL.
In one specific embodiment, in the feature map constructed by the construction method, the integration parameter is set to 6000 as the minimum peak area.
The embodiment of the invention also provides a quality detection method of the fructus alpiniae oxyphyllae formula granules, which comprises the following steps:
preparation of index component reference solution: dissolving a nootkatone reference substance in a solvent to prepare index reference substance solutions with different concentrations;
preparing a sample solution to be tested: taking fructus Alpinae Oxyphyllae formula granules to be detected, adding an extraction solvent for extraction, filtering an extracting solution, and taking a subsequent filtrate to obtain a test solution;
establishing a standard curve: respectively injecting the index component reference substance solutions with different concentrations into an ultra-high performance liquid chromatograph for detection, and constructing a concentration-peak area standard curve of the index component;
and (3) detection: injecting the sample solution to be detected into an ultra-high performance liquid chromatograph for detection, and comparing a detection map with a concentration-peak area standard curve of an index component;
wherein, the mobile phase A adopted by the ultra-high performance liquid chromatograph is acetonitrile, the mobile phase B is formic acid water solution with the volume fraction of 0.05 percent to 0.15 percent, and the elution mode is gradient elution.
Specifically, the method for preparing the sample solution to be tested is the same as the method for preparing the sample solution in the above feature map construction, and the conditions related to the ultra high performance liquid chromatography in the standard curve establishment and detection step are the same as the above feature map construction, which are not repeated herein.
In one specific embodiment, the solvent used in the preparation of the index component control solution may be an alcohol solvent, preferably methanol. More specifically, the concentrations of the index component reference solutions with different concentrations can be set to 80-90 μ g/mL and 75-85 μ g/mL.
In one specific embodiment, the quality detection method further comprises the steps of constructing a characteristic map of the fructus alpiniae oxyphyllae formula granules according to the construction method; and comparing the obtained detection map of the sample to be detected with the characteristic map. Namely, the quality detection method can realize the detection of the characteristic spectrum and the nootkatone content of the fructus alpiniae oxyphyllae formula particles, and simply, conveniently and comprehensively monitor the quality of the fructus alpiniae oxyphyllae formula particles.
The following specific examples are provided, and the raw materials used in the examples are all commercially available unless otherwise specified.
The experimental materials and instruments used in the specific examples were as follows:
experimental materials:
a round teak reference substance (6-JTN-173-1, Shenzhen Lijing);
kaempferide reference (110861-201209, China institute for testing food and drug); acetonitrile, 0.1% formic acid.
Fructus alpiniae oxyphyllae formula granules are provided by Guangdong party pharmaceutical company Limited, 10 batches in total, and the preparation method is as follows:
selecting fructus Alpinae Oxyphyllae, removing impurities to obtain fructus Alpinae Oxyphyllae decoction pieces, decocting, collecting volatile oil, and clathrating the volatile oil with beta-cyclodextrin to obtain beta-cyclodextrin clathrate; collecting residues, decocting twice, vacuum concentrating the filtrate under reduced pressure to obtain fluid extract, spray drying the fluid extract, mixing the spray dried powder and the beta-cyclodextrin inclusion compound, and dry granulating.
The batch numbers are shown in Table 1 below:
TABLE 1
The instrument comprises the following steps: ultra-high performance liquid chromatograph Waters acquisition UPLC; a chromatographic column: agilent ZORBAX SB-C18 chromatographic column, 2.1mm × 100mm, 1.8 um; XP26 millionth-one-day (METTLER TOLEOD); an ultrasonic instrument: KQ-500DE model ultrasonic cleaner (300W, 40 Hz).
Example 1
This example is a method for constructing a characteristic spectrum of a fructus alpiniae oxyphyllae formula granule.
1. Chromatographic conditions and system applicability experiments: a chromatographic column: agilent ZORBAX SB-C18 chromatographic column, 2.1mm × 100mm, 1.8 um; acetonitrile is taken as a mobile phase A, 0.1mL of formic acid solution in each 100mL of water is taken as a mobile phase B, and gradient elution is shown in a table 2; the column temperature is 30 ℃, the flow rate is 0.3ml/min, the detection wavelength is 254nm, and the theoretical plate number is not less than 100000 calculated according to the nootkatone peak.
TABLE 2
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～10 | 8～16 | 92～84 |
| 10～25 | 16～23 | 84～77 |
| 25～35 | 23～73 | 77～27 |
| 35～38 | 73～8 | 27～92 |
2. Preparation of the solution
Preparation of control solutions: taking a proper amount of the control substances of the nootkatone and the kaempferide, accurately weighing, placing the control substances into a volumetric flask, adding methanol, and preparing the concentrations of the methanol into control substance solutions of 110.3 mu g/mL and 82.92 mu g/mL respectively.
Preparation of a test solution: taking 0.1g of fructus alpiniae oxyphyllae formula particles prepared in experimental materials, grinding, precisely weighing, placing in a conical flask with a plug, adding 10mL of methanol water with 50% of volume fraction, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 25kHz) for 30 minutes, cooling, weighing again, complementing the reduced weight with methanol water with 50% of volume fraction, shaking uniformly, and filtering to obtain the fructus alpiniae oxyphyllae beverage.
3. Feature map construction
Respectively sucking 2 μ L of each of the reference solution and the sample solution, injecting into ultra high performance liquid chromatograph (Waters ACQUITY UPLC), and measuring according to "chromatographic conditions and system applicability experiment". Chromatogram of the obtained reference substances kaempferide and nootkatone are shown in FIGS. 1-2.
The 10 batches of sharpleaf galangal fruit formula granules are measured, an obtained characteristic spectrum is established and shown in figure 4, 15 characteristic peaks are shown, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, kaempferide, peak 13, nootkatone and peak 15, and the minimum peak area is set as 6000 according to integral parameters. The retention time of each characteristic peak is shown in table 3 below.
TABLE 3
4. Standard decoction and characteristic spectrum qualitative identification of fructus alpiniae oxyphyllae formula granules prepared by traditional process
4.1 preparation method of fructus Alpinae Oxyphyllae standard decoction is as follows:
a. selecting fructus Alpinae Oxyphyllae, removing impurities, removing shell, and mashing to obtain fructus Alpinae Oxyphyllae decoction pieces.
b. Decocting fructus Alpinae Oxyphyllae decoction pieces with water twice, adding 10 times of water for the first time, soaking for 40 min, decocting for 60 min, adding 8 times of water for the second time, decocting for 40 min, and collecting decoction;
c. filtering the decoction, and concentrating under reduced pressure to obtain fructus Alpinae Oxyphyllae fluid extract;
d. and (4) taking the fructus alpiniae oxyphyllae clear paste, and freeze-drying to obtain the fructus alpiniae oxyphyllae standard decoction freeze-dried powder.
4.2 the method for preparing fructus Alpinae Oxyphyllae formula granules by the traditional process is as follows:
a. selecting 6 batches of fructus alpiniae oxyphyllae medicinal materials, and removing impurities to obtain fructus alpiniae oxyphyllae decoction pieces;
b. decocting 6 batches of fructus Alpinae Oxyphyllae decoction pieces with water twice, and filtering the filtrate;
c. vacuum concentrating the filtrate under reduced pressure to obtain fructus Alpinae Oxyphyllae fluid extract;
d. spray drying fructus Alpinae Oxyphyllae fluid extract, mixing the spray dried powder, and dry granulating.
The fructus alpiniae oxyphyllae standard decoction is detected according to the characteristic map construction method of the fructus alpiniae oxyphyllae formula granules in the example 1, and the fructus alpiniae oxyphyllae standard decoction map is obtained and is shown in figure 4. Meanwhile, the fructus alpiniae oxyphyllae formula granules prepared by the conventional process are measured according to the characteristic map construction method of the fructus alpiniae oxyphyllae formula granules of example 1 in 6 batches (batch numbers are shown in table 4 below), and the characteristic map prepared by the conventional process is obtained, and is shown in fig. 5.
TABLE 4
As can be seen from the comparison between the characteristic spectrum (fig. 3) of the fructus alpiniae oxyphyllae formula particle constructed by the invention in example 1, the standard decoction spectrum (fig. 4) of fructus alpiniae oxyphyllae and the traditional preparation spectrum (fig. 5), the characteristic spectrum of the fructus alpiniae oxyphyllae formula particle constructed by the invention is consistent with the standard decoction spectrum, and compared with the traditional preparation characteristic spectrum, one more characteristic peak is formed (peak number: 15).
Example 2
This example is a method for measuring the amount of nootkatone in fructus alpiniae oxyphyllae formula granules.
1. Chromatographic conditions and system applicability experiments: a chromatographic column: agilent ZORBAX SB-C18 chromatographic column, 2.1mm × 100mm, 1.8 um; acetonitrile is taken as a mobile phase A, 0.1mL of formic acid solution in each 100mL of water is taken as a mobile phase B, and gradient elution is shown in a table 5; the column temperature is 30 ℃, the flow rate is 0.3ml/min, the detection wavelength is 254nm, and the theoretical plate number is not less than 100000 calculated according to the nootkatone peak.
TABLE 5
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～10 | 8～16 | 92～84 |
| 10～25 | 16～23 | 84～77 |
| 25～35 | 23～73 | 77～27 |
| 35～38 | 73～8 | 27～92 |
2. Preparation of the solution
Preparation of index component reference solution: approximately 5mg of nootkatone control was weighed precisely and placed in a volumetric flask, and methanol was added to prepare control solutions having concentrations of 85.6. mu.g/mL (nootkatone 1) and 78.9. mu.g/mL (nootkatone 2), respectively.
Preparation of a test solution: taking 0.1g of fructus alpiniae oxyphyllae formula particles prepared in experimental materials, grinding, precisely weighing, placing in a conical flask with a plug, adding 10mL of methanol water with 50% of volume fraction, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 25kHz) for 30 minutes, cooling, weighing again, complementing the reduced weight with methanol water with 50% of volume fraction, shaking uniformly, and filtering to obtain the fructus alpiniae oxyphyllae beverage.
3. Concentration-peak area standard curve establishment and determination of nootkatone content in sample to be measured
Respectively sucking 2 μ L of index component reference solution with different concentrations, injecting into ultra high performance liquid chromatograph (Waters ACQUITY UPLC), measuring according to "chromatographic condition and system applicability experiment", respectively injecting sample twice, and averaging. The results are shown in table 6 below.
TABLE 6 control information
According to the external standard method, a sample concentration calculation equation can be obtained: c ═ 2 (sample a mean/13778.27 + sample a mean/13869.07)/2, and then the content was calculated according to concentration: content-cxdilution factor/sample amount.
Sucking 2 μ L of sample solution, injecting into ultra high performance liquid chromatograph (Waters ACQUITY UPLC), measuring according to "chromatographic conditions and system applicability test", introducing sample twice (marked as sample 1 and sample 2), and averaging. The nootkatone content of this batch of granules was found to be 0.6979mg/g by calculation (see Table 7 below).
TABLE 7 sample content
Example 3 methodological validation
3.1 precision
3.1.1 repeatability tests: 6 portions of the same batch of fructus Alpinae Oxyphyllae formula granules (code: S3) are prepared and tested. Results the nootkatone content was found to be very reproducible and is shown in Table 8.
TABLE 8 results of the repeatability test of nootkatone
3.1.2 control precision: 6 parts of nootkatone reference substance of the same batch are taken, and the peak area is measured according to a detection method. The results show that nootkatone has good precision, as shown in Table 9.
TABLE 9 nootkatone precision
3.2 Linear relationship investigation
Respectively sucking 0.05mL, 0.1mL, 1mL, 2mL, 4mL and 6mL of the mother solution of the nootkatone reference substance, adding methanol to fix the volume to 10mL, injecting into an ultrahigh liquid chromatograph, and obtaining a regression equation by taking a peak area as a vertical coordinate, taking the concentration (ug/mL) of the reference substance as a horizontal coordinate and taking the nootkatone in the concentration range of 0.1103-66.18 ug/mL: Y14976X +749.98 (R)20.9993), the results are shown in table 10.
TABLE 10 grapefruit Ketone Linear relationship examination
3.3 stability study
The same test solution was taken and measured at different times, and the results are shown in Table 11:
TABLE 11 stability test wiper for test solutions
The peak area of nootkatone in the alpinia oxyphylla formula granules measured within 12 hours was stable, and the RSD of the nootkatone peak area was 0.62%, indicating that the test solution was stable within 12 hours.
3.4 specificity
Preparing fructus alpiniae oxyphyllae formula particle beta-cyclodextrin negative control solution: weighing 0.1g of beta-cyclodextrin, placing the beta-cyclodextrin into a conical flask with a plug, adding 10mL of methanol water with 50% of volume fraction, sealing the plug, weighing, carrying out ultrasonic treatment (250W, frequency of 25kHz) for 30 minutes, cooling, weighing again, complementing the reduced weight with the methanol water with 50% of volume fraction, shaking up, and filtering to obtain the beta-cyclodextrin negative sample solution.
Preparation of fructus alpiniae oxyphyllae formula particle silicon dioxide negative control solution: weighing 0.1g of silicon dioxide, placing the silicon dioxide in a conical flask with a plug, adding 10mL of methanol water with 50% of volume fraction, sealing the plug, weighing, carrying out ultrasonic treatment (250W, frequency 25kHz) for 30 minutes, cooling, weighing again, complementing the reduced weight with the methanol water with 50% of volume fraction, shaking up, and filtering to obtain a silicon dioxide negative sample solution.
Preparing a fructus alpiniae oxyphyllae formula particle negative control solution: taking 50% methanol by volume fraction, carrying out ultrasonic treatment (250W, frequency 25kHz) for 30 minutes, cooling, and filtering to obtain the product.
Precisely absorbing 2uL of each of the beta-cyclodextrin negative sample solution, the silicon dioxide negative sample solution and the 50% methanol negative control solution, respectively injecting into a super high liquid chromatograph, and determining according to a detection method. Results negative control solutions were not interfered with in any of the tests (see FIGS. 6, 7, 8).
3.5 sample recovery
9 parts of a same batch of test sample with known content (number: S3) are taken and respectively placed in conical flasks with stoppers, the sampling amount of each part is half of the sampling amount (0.1g) of the test sample, nootkatone with the concentration of 110.3ug/mL is precisely added according to the table 12 and the current sampling content is 1:0.5, 1:1 and 1:1.5, 50% methanol is added for constant volume to 10mL, the stoppers are sealed and the weight is weighed, the treatment and the detection are carried out according to the preparation method of the test sample, and the recovery rate of the nootkatone is calculated. As a result, the average recovery rate of nootkatone was 95.20%, and RSD was 3.39%. The specific results are shown in Table 12.
TABLE 12 test results of sample recovery of nootkatone
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. A method for identifying the characteristic spectrum of a traditional fructus alpiniae oxyphyllae formula particle, a non-traditional fructus alpiniae oxyphyllae formula particle and a fructus alpiniae oxyphyllae standard decoction is characterized by comprising the following steps:
preparation of control solutions: dissolving pomelo ketone and kaempferide reference substances in solvent to obtain reference substance solution;
preparation of a test solution: extracting conventional fructus Alpinae Oxyphyllae granule, non-conventional fructus Alpinae Oxyphyllae granule or standard decoction of fructus Alpinae Oxyphyllae with solvent, filtering the extractive solution, and collecting filtrate to obtain sample solution;
detecting the reference substance solution and the test substance solution by using an ultra-high performance liquid chromatography, wherein the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is a formic acid aqueous solution with the volume fraction of 0.05% -0.15%, and the elution mode is gradient elution;
the gradient elution included the following procedure:
the volume percentage of the mobile phase A is increased from 8% to 16% in 0-10 min;
the volume percentage of the mobile phase A is increased from 16% to 23% within 10-25 min;
the volume percentage of the mobile phase A is increased from 23% to 73% within 25-35 min;
reducing the volume percentage of the mobile phase A from 73% to 8% in 35-38 min;
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
the preparation method of the traditional fructus alpiniae oxyphyllae formula particle comprises the following steps:
a. selecting fructus Alpinae Oxyphyllae, and removing impurities to obtain fructus Alpinae Oxyphyllae decoction pieces;
b. decocting fructus Alpinae Oxyphyllae decoction pieces with water twice, and filtering the filtrate;
c. vacuum concentrating the filtrate under reduced pressure to obtain fructus Alpinae Oxyphyllae fluid extract;
d. spray drying fructus Alpinae Oxyphyllae fluid extract, mixing spray dried powder, and dry granulating;
the preparation method of the unconventional fructus alpiniae oxyphyllae formula particle comprises the following steps:
selecting fructus Alpinae Oxyphyllae, removing impurities to obtain fructus Alpinae Oxyphyllae decoction pieces, decocting, collecting volatile oil, and clathrating the volatile oil with beta-cyclodextrin to obtain beta-cyclodextrin clathrate;
collecting the residues, decocting twice, vacuum concentrating the filtrate under reduced pressure to obtain fluid extract, spray drying the fluid extract, mixing the spray dried powder with the beta-cyclodextrin inclusion compound, and dry granulating;
the preparation method of the fructus alpiniae oxyphyllae standard decoction comprises the following steps:
a. selecting fructus Alpinae Oxyphyllae, removing impurities, removing shell, and mashing to obtain fructus Alpinae Oxyphyllae decoction pieces;
b. decocting fructus Alpinae Oxyphyllae decoction pieces with water twice, adding 10 times of water for the first time, soaking for 40 min, decocting for 60 min, adding 8 times of water for the second time, decocting for 40 min, and collecting decoction;
c. filtering the decoction, and concentrating under reduced pressure to obtain fructus Alpinae Oxyphyllae fluid extract;
d. and (4) taking the fructus alpiniae oxyphyllae clear paste, and freeze-drying to obtain the fructus alpiniae oxyphyllae standard decoction freeze-dried powder.
2. The feature pattern identification method according to claim 1, wherein the conditions of the ultra high performance liquid chromatography include:
detection wavelength: 250-260 nm; flow rate: 0.25-0.35 mL/min; column temperature: 25-35 ℃.
3. The feature pattern identification method according to claim 2, wherein the conditions of the ultra high performance liquid chromatography include:
detection wavelength: 254 nm; flow rate: 0.3 mL/min; column temperature: at 30 ℃.
4. The method for identifying the feature pattern according to any one of claims 1 to 3, wherein the sample solution is prepared by using a methanol aqueous solution with a volume fraction of 45% to 55%.
5. The method for identifying a feature pattern according to claim 4, wherein the sample solution is prepared by using a methanol aqueous solution having a volume fraction of 50%.
6. The feature profile identification method according to claim 4, wherein the extraction method is ultrasonic extraction: the ultrasonic power is 200-300W, and the frequency is 20-30 kHZ; the extraction time is 25-35 min.
7. The feature spectrum identification method according to any one of claims 1 to 3, wherein a solvent used in the preparation of the control solution is an alcohol solvent.
8. The signature panel identification method of claim 7, wherein the alcohol solvent is methanol.
9. The method for identifying a characteristic spectrum according to any one of claims 1 to 3, wherein the concentration of nootkatone in the control solution is 105 to 115 μ g/mL and the concentration of kaempferide is 80 to 90 μ g/mL.
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